The human beta-globin Locus Control Region (LCR) is composed of 5 DNase I hypersensitive sites (HS) located 6-22 kb upstream to the epsilon- globin gene on chromosome 11. Naturally occurring deletions which remove the LCR result in the failure to activate all cis linked globin genes. Constructs containing all or some of these HS have been shown to produce high level erythroid specific expression in transgenic mice and transfected cells. Although the LCR contains several regulatory elements, including an erythroid specific enhancer and a nuclear matrix attachment region (MAR), how each element functions to produce a transcriptionally active, DNase I sensitive, early replicating domain in vivo is not understood. In this application, we propose to dissect the role of individual elements of the LCR in the formation of an active, early replicating and transcriptionally active chromatin domain through experiments using homologous recombination to site specifically add back or delete elements of the LCR in their normal chromosomal location. Although homologous recombination has been used to alter structural genes, this approach has not yet been used to study the function of genetic regulatory elements. Through the use of homologous recombination, we will alter regulatory elements in their native chromosomal location and determine the effects of these alterations on both the initiation and maintenance of replication timing, chromatin structure and transcriptional activity. Thus, our proposed experiments represent a novel approach to understanding the contribution of specific control elements to gene structure and function. In addition, we will also use the more conventional transgenic and transfection approaches to complement the homologous recombination analyses. Our specific aims include: (1) accurate mapping of the human beta-globin domain DNase I sensitive and replication domains; (2) determining if and which MAR/enhancer combinations generate a red-cell specific LCR, as measured in transgenic and transfection assays; (3)determining the structural and functional consequences of LCR element additions or deletions in their native chromosomal location by performing homologous recombination experiments to alter mutant and normal beta-globin loci in committed mouse erythroid (MEL) cell hybrids; (4) determining the role of individual elements in the initiation of an active beta-globin domain by performing homologous recombination experiments in non-erythroid cells prior to fusion with Mel cells or in embryonic stem (ES) cells; and (5) determining the role of the LCR in establishing an early replicating, active chromatin structure by analyzing the chromatin structure and replication of mouse DNA at sites of integration of LCR cassettes in transfected MEL cells.